The repeated use of drugs of abuse produce long term alterations in behavior and brain function that contribute to the compulsive drug-seeking that characterizes addiction. Research conducted in the Integrative Neuroscience Section seeks to identify the neural substrates that lead to these adaptive responses as well as those endogenous systems within the brain that oppose their development and long-term expression. The long-term goal of these studies is to identify effective targets for the treatment of drug and alcohol addiction and biological factors that may result in increased vulnerability to acquire compulsive drug seeking behavior. Our studies have continued to focus on endogenous opioid systems since we have demonstrated important and contrasting roles of opioid peptides (dynorphin, endorphin, enkephalin and their receptors (mu-, kappa- and delta-) in regulating circuits in the brain upon which drugs of abuse acts to produce their incentive motivational effects. Using in-vivo neurochemical and intracellular recordings, we have begun to identify the role of these systems in regulating neurotransmission in brain regions comprising the extended amygdala. We have found that kappa-opioid receptors inhibit dopaminergic neurons that project to the medial prefrontal cortex but do not modulate the activity of neurons projecting to the nucleus accumbens. This in turn results in decreased dopamine release in the prefrontal cortex but not in the accumbens. Our studies have also identified a population of GABAergic neurons that project to the prefrontal cortex and are inhibited by mu-opioid receptors. Ongoing studies are examining the role of mu- and kappa ?opioid systems in regulating dopamine, GABA and glutamate neurotransmission in this brain regions as well as the central and basolateral amygdala.We have continued our studies examining the role of endogenous kappa-opioid receptor systems in mediating individual differences in susceptibility to cocaine and other drugs of abuse. We have shown that a decrease in the activity of this opioid system enhances the cellular, neurochemical and behavioral effects produced by acute exposure to cocaine. Furthermore, individuals in which this opioid system is inactivated also exhibit an enhanced responsiveness to the acute effects of alcohol. Building on our previous demonstration that kappa-opioid receptors regulate the activity of the dopamine transporter, a membrane protein upon which cocaine and other psychostimulants act to exert their pharmacological effects, we have initiated studies to determine the intracellular mechanisms mediating this effect. Using the technique of bioluminescence resonance energy transfer (BRET), we have obtained evidence that kappa-opioid receptors dimerize with dopamine transporters and that a physical association of these two proteins may contribute to the ability of this opioid receptor subtype to regulate dopamine transporter function and the effects of cocaine. In view of these data, we have initiated studies to determine whether other G-protein coupled receptors that are located in the brain reward circuit can regulate biogenic amine transporter function and identify the mechanisms of this effect. To accomplish these goals, we have developed and validated a fluorescence imaging approach that permits time-resolved quantification of serotonin, norepinephrine and dopamine transporter function in individual living cells. We have obtained the first unequivocal evidence that D3 receptors up regulate dopamine transporter function in heterologous and neural cell lines. Our studies have also revealed that D3 receptors activate mitogen activate protein kinase and phosphoinositol-3 kinase and that these actions are required for regulation of transport. These findings are noteworthy in that they provide a novel mechanism by which D3 receptors which are located presynaptically on mesolimbic dopamine neurons regulate dopamine neurotransmission. Importantly, we have also demonstrated a novel mechanism by which D3 receptors modulate the effects of amphetamine. Using immunofluorescence confocal microscopy and biochemical techniques that permit quantification of transporter trafficking from the membrane to the cytosol, we have shown that D3 receptor activation prevents the internalization of dopamine transporters produced by amphetamine. Such findings have important clinical implications, in view of recent work, which suggests that targeting of D3 receptors may be effective in the treatment of addiction, schizophrenia and Parkinson?s disease.